Methods for fabricating polymer-based controlled release devices

ABSTRACT

The present invention relates to a polymer-based sustained release device, and methods of forming and using the device for the sustained release of an active agent. The improved method of the invention for forming a polymer-bases sustained release device comprises forming a polymer/active agent solution by mixing a polymer, a continuous phase, and an active agent. The continuous phase can comprise one or more polymer solvents, a polymer solvent/polymer non-solvent mixture, or a polymer solvent/active agent non-solvent mixture. When the continuous phase comprises a polymer solvent/active agent non-solvent, the active agent can also be present as a microparticulate rather than in solution. The continuous phase is then removed from the polymer/active agent solution, thereby forming a solid polymer/active agent matrix.

BACKGROUND

Many illnesses or conditions require administration of a constant orsustained level of a medicament or biologically active agent to providethe most effective prophylactic or therapeutic effect. This may beaccomplished through a multiple dosing regimen or by employing a systemthat releases the medicament in a sustained fashion.

Systems for delivering sustained levels of medication have employedbiodegradable materials, such as polymers, encapsulating the medicament.The use of biodegradable polymers, for example, in the form ofmicroparticles or microcarriers, provides a sustained release ofmedicaments, by utilizing the inherent biodegradability of the polymerto control the release of the medicament thereby providing a moreconsistent, sustained level of medication and improved patientcompliance.

Certain methods of fabricating polymer-based sustained release devicescomprise the steps of dissolving a polymer in a solvent, adding to thepolymer solution the active agent to be incorporated and removing thesolvent from the mixture thereby forming a matrix of the polymer withthe active agent distributed throughout the matrix.

Many of these methods of fabricating polymer-based sustained releasedevices employ a solvent or mixture of solvents, which solubilizes thepolymer, but are not capable of solubilizing the active agent to beincorporated. Hence, these methods have disadvantages, for example, inthe lack of suitable solvents which are capable of dissolving bothactive agent and polymer and which are non-toxic, biocompatible and canbe readily removed from the final product; in solubilizing of the activeagent in an active form; and in optimizing encapsulation efficiency ofthe active agent to achieve a device with the desired releasecharacteristics.

Therefore, a need exists for improved methods for fabricating apolymer-based sustained release device, particularly devices having ahigh load of active agent.

SUMMARY OF THE INVENTION

The present invention is based upon the discovery that an improvedpolymer-based sustained release device can be achieved when a continuousphase which is capable of solubilizing both the polymer and the activeagent is employed in the method for fabricating the device.Unexpectedly, an advantage of the sustained release devices obtainedthereby is that they can have a very high load of active agent. Forexample, the device can achieve a relative weight of active agent inexcess of the total polymer weight (e.g., present at about 50% by weightor more of the total weight of the device) with improved encapsulationefficiency and improved sustained release characteristics.

An additional advantage of the invention is that it allows for thepreparation of small microparticles which contain encapsulated drug andexhibit improved delivery characteristics. A further advantage is theability to use solubility properties of the active agent to affectparticle size of the active agent, further enabling improved deliverycharacteristics. Additionally, the process for preparing microparticlesmay be improved by permitting the ability to filter sterilize processcomponents or facilitate atomization of the polymer/active agentsolution or dispersion.

The present invention thus relates to a polymer-based sustained releasedevice, and methods of forming and using said device for the sustainedrelease of an active agent. The improved method of the invention, forforming the polymer-based sustained release device, utilizes acontinuous phase which comprises, for example, one or more polymersolvents, a polymer solvent/polymer non-solvent mixture or a polymersolvent/active agent non-solvent mixture, to dissolve the polymer andalso solubilize the active agent in the polymer solution. Also embracedby the invention described herein is a process wherein the continuousphase comprises a polymer solvent/active agent non-solvent mixture andthe active agent is present as a microparticulate. For purposes of theinvention, the term "microparticulate" describes the situation where theactive agent is dispersed in the continuous phase at a concentration ofthe active agent approaching solubilization of the active agent or wherethe active agent is present as a combination of both dispersedparticulate and solubilized active agent. Typically, themicroparticulate is formed by mixing an active agent non-solvent with asolution containing the active agent which thereby leads to partial orcomplete precipitation of the active agent (also referred to as the"Microparticulate Method").

In one embodiment, the method comprises (a) forming a polymer/activeagent solution by mixing a polymer, a continuous phase comprising one ormore polymer solvents and an active agent wherein the polymer and activeagent are present in relative concentrations such that the final productcontains about 50% by weight or more of active agent; and (b) removingthe continuous phase of step (a) thereby forming a solid polymer/activeagent matrix.

The method can further comprise the step of forming droplets of thepolymer/active agent solution prior to removal of the continuous phase.Further, the method can comprise freezing the droplets prior to removalof the continuous phase. According to the method of the invention thedroplets can be microdroplets. In a specific embodiment wherein dropletsare formed and then frozen, the continuous phase can be removed by anextraction process. Alternatively, the continuous phase can be removedby an evaporation process or a combination of an evaporation andextraction process.

When the continuous phase comprises one or more polymer solvents anycombination of polymer solvents which is miscible and allows both thepolymer and active agent to be dissolved, is suitable for use in theinvention. Dimethylsulfoxide (also referred to as DMSO) is preferredbecause it is a good solvent for many polymers and active agents,including water-soluble agents such as peptides, antigens, and smallmolecule drugs. Other suitable solvents, in particular for PLGA polymersinclude, DMSO, ethyl acetate, methyl acetate, methylene chloride,chloroform, hexafluoroisopropanol, acetone, and combinations thereof.Preferably, the polymer solvent is pharmaceutically acceptable.

In another embodiment, the method for forming a polymer-based sustainedrelease device comprises the steps of: (a) forming a polymer/activeagent solution by mixing a polymer, an effective amount of an activeagent and a continuous phase comprising a polymer solvent/polymernon-solvent mixture wherein the amount of polymer non-solvent isdictated by achieving solubilization of the active agent without causingsubstantial precipitation of the polymer; and (b) removing thecontinuous phase of step (a) from the polymer/active agent solution,thereby forming a solid polymer/active agent matrix. In a furtherembodiment, the active agent is present at a concentration such that thefinal product or device contains about 50% by weight or more of activeagent.

The method can further comprise the step of forming droplets of thepolymer/active agent solution prior to removal of the continuous phase.Further, the method can comprise freezing the droplets prior to removalof the continuous phase. According to the method of the invention thedroplets can be microdroplets. In a specific embodiment wherein dropletsare formed and then frozen, the continuous phase can be removed by anextraction process. Alternatively, the continuous phase can be removedby evaporation process or a combination of an evaporation and extractionprocess.

The polymer non-solvent can be selected such that it is miscible withthe polymer solvent, does not cause substantial precipitation of thepolymer and is not deleterious to the active agent. Preferably, thepolymer solvent and the polymer non-solvent are pharmaceuticallyacceptable.

Suitable polymer non-solvents include, for example, ethanol, methanol,water, acetonitrile (MeCN), dimethylformamide (DMF), ethyl ether,alkanes such as pentane, isopentane, hexane, heptane and oils, such asmineral oils, fish oils, silicone oils, vegetable oils, or combinationsthereof. Vegetable oils, such as olive oil, sesame oil, soybean oil,safflower oil, peanut oil, cottonseed oil, coconut oil, linseed oil,corn oil, castor oil, palm oil, or combinations thereof, are preferredfor use in the invention. In particular embodiments, the polymer solventis DMSO and the non-solvent is ethanol or water.

In another embodiment, the method for forming a polymer-based sustainedrelease device comprises the steps of: (a) forming a polymer/activeagent mixture by mixing a polymer, an effective amount of an activeagent and a continuous phase comprising a polymer solvent/active agentnon-solvent mixture wherein the amount of active agent non-solvent isdictated by achieving solubilization of the active agent, oralternatively achieving the active agent as a microparticulate in thecontinuous phase containing the polymer; and (b) removing the continuousphase of step (a) thereby forming a solid polymer/active agent matrix.In a further embodiment, the active agent is present at a concentrationsuch that the final product or device contains about 50% by weight ormore of active agent.

The method can further comprise the steps of forming droplets of thepolymer/active agent solution prior to removal of the continuous phase.Further, the method can comprise freezing the droplets prior to removalof the continuous phase. According to the method of the invention thedroplets can be microdroplets. In a specific embodiment wherein dropletsare formed and then frozen, the continuous phase can be removed by anextraction process. Alternatively, the continuous phase can be removedby evaporation process or a combination of an evaporation and extractionprocess.

The active agent non-solvent can be selected such that it is misciblewith the polymer solvent, does not substantially precipitate thepolymer, and is not deleterious to the active agent. Suitable activeagent non-solvents are dependent upon the properties of the active agentand for peptides can include, for example, acetone, ethanol andmethylene chloride.

In another aspect, the invention relates to a polymer-based sustainedrelease device prepared according to the method of the invention. Thedevice comprises a polymeric matrix and an active agent dispersed withinthe matrix. The device formed by the method of the invention exhibits aunique microstructure, the porosity of which varies as a function ofload, polymer concentration and the type of continuous phase employed.

The method of using the polymer-based sustained release device of thepresent invention comprises providing a sustained delivery of activeagent, in a subject, over a therapeutically useful period of time, byadministering to the subject a dose of said polymer-based sustainedrelease device. The invention also provides methods for preparingmicroparticles of varying size and/or morphology for use in specificapplications, for example, applications such as chemoembolization,vaccine delivery or cellular uptake where the size of the microparticlesdirectly impacts Performance.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plot of the percent of animals per treatment group indiestrus for groups of rats treated with microparticles prepared usingthe Particulate Method, as described herein, and having the indicatedload of azaline B versus time.

FIG. 2 is a plot of the percent of animals per treatment group indiestrus for groups of rats treated with microparticles having theindicated load of azaline B prepared using the method of the invention,according to Examples 1 and 3, as described herein, and having theindicated load of azaline B, versus time.

FIG. 3 is a plot of serum concentrations (ng/ml) of azaline B for groupsof animals treated with azaline B containing microparticles prepared byusing the Particulate Method and the method of the invention, accordingto Examples 1 and 3, as described herein, and having the indicated loadof azaline B, versus time.

DETAILED DESCRIPTION OF THE INVENTION

The features and other details of the invention will now be moreparticularly described and pointed out here as well as in the claims. Itwill be understood that the particular embodiments of the invention areshown by way of illustration and not as limitations of the invention.The principle features of this invention can be employed in variousembodiments without departing from the scope of the invention.

A solution, as defined herein, is a mixture of one or more substances(referred to as the solute, for example, the polymer and the activeagent) dissolved in one or more other substances (referred to as thesolvent or solvents, for example, DMSO or a combination of DMSO andmethylene chloride). For purposes of this invention, the "continuousphase" refers to the major component of a solution, such as a polymersolvent or a mixture thereof, and a mixture of a polymer solvent and anon-solvent.

The term "non-solvent," as used herein, refers to a material, which doesnot substantially dissolve a second or reference material. For purposesof this invention, the non-solvent can be a non-solvent for the activeagent or the polymer.

The term "microdroplet," as used herein, refers to a droplet of anymorphology which has a dimension less than or equal to about 1000 μm.

The active agent, azaline B, used in many of the examples describedherein is an LHRH peptide analog, the structure of which is describedin, for example, Campen et al., Biochemical Pharmacology 40: 1313-1321,1995, and which can be depicted as follows: Ac--D-Nal¹ -D-Cpa² -D-Pal³-Ser⁴ -Aph⁵ (atz)-D--Aph⁶ (atz)-Leu⁷ -Ilys⁸ -Pro⁹ -D Ala¹⁰ (Ac=acetyl,Nal=3-(2'-naphthyl)-alanine, Cpa=4-chloro-phenyalanine,Pal=3-(3'-pyridyl)-alanine, Aph=4-amino-phenylalanine,atz=5'-(3'-amino-1H-1', 2', 4'-triazolyl), Ilys=N'-isopropyl-lysine).The azaline B can also be in the form of salt, such as the acetate salt.

In one aspect, the invention provides an improved method for preparing apolymer-based sustained release device comprising the use of acontinuous phase which comprises one or more polymer solvents, a mixtureof one or more polymer solvents with one more polymer non-solvents or amixture of one or more polymer solvents with one or more active agentnon-solvents, to dissolve the polymer and also solubilize the activeagent in the polymer solution. When the continuous phase comprises apolymer solvent/active agent non-solvent, the situation where the activeagent is present as a microparticulate is also embraced within theinvention described herein.

In one embodiment, the method comprises (a) forming a polymer/activeagent solution by mixing a polymer, a continuous phase comprising one ormore polymer solvents and an active agent wherein polymer and activeagent are present in relative concentrations such that the final productor device contains about 50% by weight or more of active agent; and (b)removing the continuous phase of step (a) thereby forming a solidpolymer/active agent matrix.

The method can further comprise the step of forming droplets of thepolymer/active agent solution prior to removal of the continuous phase.Further the method can comprise freezing the droplets prior to removalof the continuous phase. According to the method of the invention thedroplets can be microdroplets. In a specific embodiment wherein dropletsare formed and then frozen, the continuous phase can be removed by anextraction process. Alternatively, the continuous phase can be removedby evaporation process or a combination of an extraction and evaporationprocess.

When the continuous phase comprises one or more polymer solvents anycombination of polymer solvents which is miscible and allows both thepolymer and active agent to be dissolved, is suitable for use in theinvention. Dimethylsulfoxide (also referred to as DMSO) is a preferredsolvent because it is a good solvent for many polymers and activeagents, including peptides, antigens and small molecule drugs. Othersuitable solvents, in particular for PLGA polymers, include, forexample, DMSO, ethyl acetate, methyl acetate, methylene chloride,chloroform, hexafluoroisopropanol and acetone. Preferably, the polymersolvent is pharmaceutically acceptable.

The method wherein one or more polymer solvents can be used as thecontinuous phase can be referred to herein as the "Polymer SolventMethod" indicating that the major component of the continuous phase ofthe method comprises, or consists essentially of, one or more polymersolvents. If more than one polymer solvent is employed, it is understoodthat one of the polymer solvents can also be a non-solvent for theactive agent provided that the active agent remains soluble in thecontinuous phase.

In another embodiment, the method for forming a polymer-based sustainedrelease device comprises the steps of: (a) forming a polymer/activeagent solution by mixing a polymer, an effective amount of an activeagent and a continuous phase comprising a polymer solvent/polymernon-solvent mixture wherein the amount of non-solvent is dictated byachieving solubilization of the active agent without causing substantialprecipitation of the polymer; and (b) removing the continuous phase ofstep (a) from the polymer/active agent solution, thereby forming a solidpolymer/active agent matrix. In a further embodiment, the active agentis present at a concentration such that the final product or devicecontains about 50% by weight or more active agent.

The method can further comprise the step of forming droplets of thepolymer/active agent solution prior to removal of the continuous phase.Further, the method can comprise freezing the droplets prior to removalof the continuous phase. According to the method of the invention thedroplets can be microdroplets. In a specific embodiment wherein dropletsare formed and then frozen, the continuous phase can be removed by anextraction process. Alternatively, the continuous phase can be removedby evaporation process or a combination of an extraction and evaporationprocess.

The method wherein the continuous phase comprises a polymersolvent/polymer non-solvent mixture, can be referred to as the "PolymerSolvent/Polymer Non-Solvent Method." When the major component of thecontinuous phase comprises, or consists essentially of, a polymersolvent/polymer non-solvent mixture a combination or one or more polymersolvents with one or more polymer non-solvents can be employed. Theamount and type of polymer non-solvent can be selected such that it iscompletely or substantially miscible with the polymer solvent, does notcause substantial precipitation of the polymer, and is not deleteriousto the active agent. Preferably, the polymer solvent and the polymernon-solvent are pharmaceutically acceptable. It is understood that oneor both solvents in the continuous phase can serve to solubilize theactive agent.

Polymer non-solvents suitable for use in the invention include, forexample, ethanol, methanol, water, acetonitrile (MeCN),dimethylformamide (DMF), ethyl ether, alkanes, such as pentane,isopentane, hexane or heptane, and oils, such as mineral oils, fishoils, silicone oil, vegetable oils, or any combination thereof.Vegetable oils, such as olive oil, sesame oil, soybean oil, saffloweroil, peanut oil, cottonseed oil, coconut oil, linseed oil, corn oil,castor oil, palm oil, or combinations thereof, are preferred for use inthe invention. In particular embodiments, the polymer solvent is DMSOand the non-solvent is ethanol or water.

In another embodiment, the method for forming a polymer-based sustainedrelease device comprises the steps of: (a) forming a polymer/activeagent mixture by mixing a polymer, an effective amount of an activeagent and a continuous phase comprising a polymer solvent/active agentnon-solvent mixture wherein the amount of active agent non-solvent isdictated by achieving solubilization of the active agent, oralternatively achieving the active agent as a microparticulate, in thecontinuous phase containing the polymer; and (b) removing the continuousphase of step (a) thereby forming a solid polymer/active agent matrix.In a further embodiment, the active agent is present at a concentrationsuch that the final product or device contains about 50% by weight ormore of active agent.

The method can further comprise the step of forming droplets of thepolymer/active agent solution prior to removal of the continuous phase.Further, the method can comprise freezing the droplets prior to removalof the continuous phase. According to the method of the invention thedroplets can be microdroplets. In a specific embodiment wherein thedroplets are formed and then frozen, the continuous phase can be removedby an extraction process. Alternatively, the continuous phase can beremoved by an evaporation process or a combination of an extraction andevaporation process.

The amount and type of active agent non-solvent can be selected suchthat it is miscible with the polymer solvent, does not substantiallyprecipitate the polymer, and is not deleterious to the active agent.Suitable active agent non-solvents are dependent upon the properties ofthe active agent and for peptides can include, for example, acetone,ethanol and methylene chloride.

Other excipients can be present in the polymer/active agent solution, asdescribed below. These excipients need not be soluble in the continuousphase, although, this is preferred.

The active agent of the invention can be added either as a solid (suchas in a fine powder) or neat liquid, or as a solution of the activeagent in the polymer solvent or polymer non-solvent.

It can be desirable to add a polymer non-solvent which is a solvent forthe active agent to the polymer solvent when forming the polymer/activeagent solution if, for example, the polymer solvent does not solubilizethe active agent to the desired degree. The polymer non-solvent shouldbe miscible with the polymer solvent, aid in solubilizing the activeagent, not cause substantial precipitation of the polymer and not bedeleterious to the active agent. An example of such an embodiment is inthe formation of a solution of PLGA and tRNA. DMSO is a good solvent forPLGA but poorly solubilizes tRNA. The inclusion of, for example, water(a good solvent for tRNA, miscible with DMSO and a non-solvent for thepolymer) results in an optically transparent solution comprising PLGA,tRNA, DMSO and water. Therefore, the continuous phase, in thisembodiment, comprises a polymer solvent/non-solvent mixture, wherein thenon-solvent is a non-solvent for the polymer. The amount of polymernon-solvent included is at least that amount necessary to achieve thedesired level of solubilization of the active agent but not to exceedthat amount which causes substantial precipitation of the polymer.

It can also be desirable to add a polymer non-solvent, which is anon-solvent for the active agent, to the polymer solvent when formingthe polymer/active agent solution, if, for example, the polymer solventsolubilizes the active agent to a greater degree than desired. Forexample, in such an embodiment, the active agent may "leach"out of themicrodroplet with the polymer solvent during the extraction step of theprocess. The addition of the active agent non-solvent can minimize thiseffect. The polymer non-solvent should be miscible with the polymersolvent and assist in decreasing the solubility of the active agent inthe resulting polymer solvent/non-solvent mixture.

In summary, one aspect of the invention relates to maximizing polymerand active agent solubility properties in the continuous phase byselecting the appropriate solvent or combination of solvents. Thus, theaddition or selection of appropriate solvents, co-solvents, ornon-solvents results in the improved microparticles described herein.

The continuous phase can be formed prior to, following or simultaneouswith the addition of the polymer to the polymer solvent. The activeagent can be mixed with the polymer solution either as a solid, a neatliquid or in solution. When the active agent is added in solution thesolvent of the active agent solution can be a polymer non-solvent,polymer solvent or combinations thereof. Further, when the active agentis added as a solid or neat liquid, which is not soluble in the polymersolution, an additional polymer solvent, polymer non-solvent orcombinations thereof can be added which solubilizes the active agent.

For example, poly(lactide-co-glycolide) was dissolved in DMSO and theactive agent, ovalbumin, was predissolved in a minimum amount of water(a polymer non-solvent) and added to the polymer solution to form thepolymer/active agent solution, thereby providing a continuous phasecomprising a polymer solvent/polymer non-solvent mixture.

In another example, poly(lactide-co-glycolide) was dissolved in DMSO andthe active agent, tRNA, was predissolved in a minimum amount of waterand added to the polymer solution to form the polymer/active agentsolution.

In yet another embodiment, the active agent can be added as a solid, toa mixture of polymer solvent/polymer non-solvent having the polymerdissolved therein. The solid is soluble in the mixture. In a specificembodiment, the continuous phase comprising the polymer solvent/polymernon-solvent mixture, is DMSO and ethanol. In a more specific embodiment,the polymer of the polymer solution includes poly(lactide-co-glycolide)dissolved in a DMSO/ethanol mixture and the active agent is azaline B.In each of these embodiments, the result was a single continuous phasein which both the polymer and active agent were solubilized, therebyavoiding prior art processes which are characterized by two or morephases. The solvents and/or non-solvents can be added in a wide range ofrelative amounts, including for example about 1:10 to about 10:1 orabout 1:3 to about 3:1, by volume, as is appropriate.

After the polymer/active agent solution is formed it can be processed toform microdroplets. These microdroplets can then be frozen by meanssuitable to form microparticles. Examples of means for processing thepolymer/active agent solution to form droplets include directing thesolution through an ultrasonic nozzle, pressure nozzle, Rayleigh jet, orby other means known for creating droplets from solution such as thosedescribed in U.S. Pat. No. 5,019,400, issued to Gombotz et al.,co-pending U.S. patent application Ser. No. 08/443,726, filed May 18,1995, and co-pending U.S. patent application Ser. No. 08/649,128, filedMay 14, 1996, the teachings of all of which are incorporated herein byreference in their entirety.

The microdroplets are then frozen by means suitable to formmicroparticles. Means suitable for freezing droplets to formmicroparticles include directing the droplets into or near a liquifiedgas, such as liquid argon and liquid nitrogen to form frozenmicrodroplets which are then separated from the liquid gas. The frozenmicrodroplets are then exposed to an extraction solvent or curingsolvent or phase, which is generally a poor solvent for the polymer,which has a lower melting point than the continuous phase and which hassufficient miscibility with the continuous phase to extract solid and/orthawed continuous phase from a frozen microparticle.

In one method the liquified gas overlays frozen extraction solvent, asdescribed in U.S. Pat. No. 5,019,400, issued to Gombotz et al., theentire content of which is incorporated herein by reference. In a secondmethod, the liquified gas and cold extraction solvent are maintained ina distinct "freezing zone" and "extraction zone," as described inco-pending U.S. patent application Ser. No. 08/443,726, filed May 18,1995, the content of which is incorporated herein in its entirety. Asstated above, the purpose of the extraction solvent is to remove orextract, as solid and/or a liquid, any continuous phase in the frozenmicrodroplets, thereby forming active agent containing microparticles.Typically, the extraction solvent or curing phase is selected from oneor more of the polymer non-solvents discussed above. It can be the sameor different as any polymer non-solvent employed in the continuousphase, as described herein. It can optionally further include an activeagent non-solvent as well. Typical extraction solvents include, forexample, ethanol, methanol, ethyl ether, alkanes such as pentane,isopentane, hexane, heptane, and oils such as mineral oils, fish oils,silicone oil, vegetable oils, or combinations thereof. Vegetable oils,such as olive oil, sesame oil, soybean oil, safflower oil, peanut oil,cottonseed oil, coconut oil, palm oil or combinations thereof, arepreferred. It is generally desirable that the extraction solvent(s) orcuring phase possess a melting point the same or, preferably, lower thanthe continuous phase. Thus, the extraction step can be conducted orinitiated at a temperature at which the extraction solvent(s) or curingphase is a liquid and the microdroplets (including the continuous phase)are frozen. Mixing ethanol with other suitable extraction solvents, suchas an alkane, including hexane, heptane or pentane, can directly impactthe encapsulation efficiency, morphology, and consistency therein of theresulting microspheres as well as cause an unexpected increase in therate of solvent extraction, above that achieved by ethanol alone, fromcertain polymers, such as poly(lactide-co-glycolide) polymers.

In another method the polymer/active agent matrix, as formed above, isfragmented at a temperature below the glass transition temperature ofthe polymer/active agent matrix, thereby forming polymer/active agentmicroparticles, as described in co-pending U.S. patent application Ser.No. 08/649,128, filed May 14, 1996 the entire teachings of which areincorporated herein by reference in their entirety.

A wide range of sizes of polymer-based sustained release devices can bemade by varying the droplet size, for example, by changing theultrasonic nozzle frequency or diameter. If larger devices are desired,the polymer/active agent solution can be processed by passage through asyringe or the like directly into the cold liquid. Alternatively, thesolution can be dripped or otherwise added to the cold liquid.Increasing the viscosity of the polymer/active agent solution can alsoincrease device size. The size of the devices which can be produced bythe method of the invention are, for example, microparticles rangingfrom greater than about 1000 to about 1 micrometer in diameter.

The term "polymer-based sustained release device," as defined herein,comprises a polymer and an active agent (also referred to herein as a"polymer/active agent matrix"). The polymers of the present inventionare generally biocompatible. Suitable biocompatible polymers can beeither biodegradable or non-biodegradable polymers, or blends orcopolymers thereof.

A polymer is biocompatible if the polymer, and any degradation productsof the polymer, are substantially non-toxic to the recipient, and alsopresents no unacceptable, deleterious or untoward effects on therecipient's body, such as a significant immunological reaction at thesite of administration.

Biodegradable, as defined herein, means the composition will degrade orerode in vivo to form smaller chemical species which arebiometabolizable and/or excretable. Degradation can result, for example,by enzymatic, chemical and/or physical processes. Suitablebiocompatible, biodegradable polymers include, for example,poly(lactides), poly(glycolides), poly(lactide-co-glycolides),poly(lactic acid)s, poly(glycolic acid)s, poly(lactic acid-co-glycolicacid)s, polycaprolactone, polycarbonates, polyesteramides,polyanhydrides, poly(amino acids), polyorthoesters, polyacetals,polycyanoacrylates, polyetheresters, poly(dioxanone)s, poly(alkylenealkylates)s, copolymers of polyethylene glycol and polyorthoester,biodegradable polyurethanes, blends and copolymers thereof.

Biocompatible, non-biodegradable polymers suitable for a sustainedrelease device include non-biodegradable polymers selected from thegroup consisting of polyacrylates, polymers of ethylene-vinyl acetatesand acyl substituted cellulose acetates, non-degradable polyurethanes,polystyrenes, polyvinyl chloride, polyvinyl fluoride, poly(vinylimidazole), chlorosulphonate polyolefins, polyethylene oxide, blends andcopolymers thereof.

Further, the terminal functionalities or pendant groups of the polymerscan be modified, for example, to modify hydrophilicity, hydrophobicityand/or provide, remove or block moieties which can interact with theactive agent (via, for example, ionic or hydrogen bonding).

Acceptable molecular weights for polymers used in this invention can bedetermined by a person of ordinary skill in the art taking intoconsideration factors such as the desired polymer degradation rate,physical properties such as mechanical strength, and rate of dissolutionof polymer in solvent. Typically, an acceptable range of molecularweights is between about 2,000 Daltons to about 2,000,000 Daltons. In apreferred embodiment, the polymer is a biodegradable polymer orcopolymer. In a more preferred embodiment, the polymer is apoly(lactide-co-glycolide) (hereinafter "PLGA").

The term "active agent," as defined herein, is an agent, or itspharmaceutically acceptable salt, which when released in vivo, possessesthe desired biological activity, for example therapeutic, diagnosticand/or prophylactic properties in vivo. Examples of suitablebiologically active agents include proteins such as immunoglobulins,antibodies, cytokines (e.g., lymphokines, monokines, chemokines),interleukins, interferons, erythropoietin, nucleases, tumor necrosisfactor, colony stimulating factors, insulin, enzymes (e.g. superoxidedismutase, a plasminogen activator), tumor suppressors, blood proteins,hormones and hormone analogs (e.g., growth hormone, adrenocorticotropichormone, luteinizing hormone releasing hormone (LHRH) and azaline B),vaccines (e.g., tumoral, bacterial and viral antigens), antigens, bloodcoagulation factors; growth factors; peptides such as proteininhibitors, protein antagonists, and protein agonists; nucleic acids,such as antisense molecules; oligonucleotides; and ribozymes. Smallmolecular weight agents suitable for use in the invention include,antitumor agents such as bleomycin hydrochloride, methotrexate andadriamycin; antibiotics such as gentamicin, tetracycline hydrochlorideand ampicillin; antipyretic, analgesic and anti-inflammatory agents;antitussives and expectorants such as ephedrine hydrochloride,methylephedrine hydrochloride, noscapine hydrochloride and codeinephosphate; sedatives such as chlorpromazine hydrochloride,prochlorperazine hydrochloride and atropine sulfate; muscle relaxantssuch as tubocurarine chloride; antiepileptics such as sodium phenytoinand ethosuximide; antiulcer agents such as metoclopramide;antidepressants such as clomipramine; antiallergic agents such asdiphenhydramine; cardiotonics such as theophillol; antiarrhythmic agentssuch as propranolol hydrochloride; vasodilators such as diltiazemhydrochloride and bamethan sulfate; hypotensive diuretics such aspentolinium and ecarazine hydrochloride; antidiuretic agents such asmetformin; anticoagulants such as sodium citrate and sodium heparin;hemostatic agents such as thrombin, menadione sodium bisulfite andacetomenaphthone; antituberculous agents such as isoniazide andethanbutol; hormones such as prednisolone sodium phosphate andmethimazole; and narcotic antagonists such as nalorphine hydrochloride.

The amount of active agent which is contained in the polymer-basedsustained release device is a therapeutically or prophylacticallyeffective amount which can be determined by a person of ordinary skillin the art taking into consideration factors such as body weight,condition to be treated, type of device used, and release rate from thedevice.

A polymeric drug delivery device of the invention can contain from about0.01% (w/w) to about 90% (w/w) of active agent (total weight ofpolymer/active agent). The amount of agent can vary depending upon thedesired effect of the agent, the planned release levels, and the timespan over which the agent is to be released. A low range of agentloading can be from about 0.1% (w/w) to about 30% (w/w). In treatmentswhere a low range of agent loading is desired a preferred range is fromabout 0.5% (w/w) to about 20% (w/w). A high range of agent loading isthat greater than or equal to about 50%. In treatments where a highrange of agent loading is employed a preferred range is from about 50%(w/w) to about 85% (w/w) and more preferably from about 50% (w/w) toabout 70% (w/w).

A sustained release of active agent is a release which occurs over aperiod of time longer than that which would be obtained followingsimilar administration of the active agent as a dispersion or solutionin a carrier. Generally, the sustained release device can deliver theactive agent for at least about seven days and, preferably, up to aboutthree months.

The polymer-based sustained release device of this invention can beformed into many shapes such as a film, a pellet, a cylinder, a wafer, adisc or a microparticle. A microparticle, generally has a diameter ofless than about one millimeter. A microparticle can have a generallyspherical, non-spherical or irregular shape. Typically, themicroparticle will be of a size suitable for injection. A preferred sizerange for microparticles is from about 1 to about 250 microns indiameter. The sustained release device in the form of a wafer or disc,for example, will typically be of a size suitable for implantation and,for example, can be manufactured by compressing microparticles.

The present invention can be used to incorporate and deliver a widevariety of active agents. Most often, the composition of the presentinvention will be used to deliver an active agent to a human or otheranimal for purposes of therapy, prophylaxis, hygiene, analgesics,cosmetics or the like. Such uses where the compositions are delivered toa human or other animal will generally be referred to as in vivo uses.The composition of the present invention will also have in vitro useswhere an active substance is being delivered to an environment or systemother than a human or animal such as in the sustained release ofagrochemicals or in diagnostics. One of the major in vivo uses for thecomposition of the present invention will be for the delivery of drugsand other pharmaceutical agents in human and veterinary applications.For both in vivo and in vitro uses, the compositions will deliver theactive substance to a surrounding environment.

Unexpectedly, the above process resulted in the ability to formsustained delivery devices even at very high loads (greater than orequal to at least about 50% (w/w)) with improved release characteristicsand duration of release, as illustrated, for example, in FIGS. 1 and 2.It was also found that the morphology of the device changed with theamount, or load, of the active agent. The device, or microparticle, wasporous at low loads (e.g, 10% to 30%), similar to the microparticlesobtained in the known processes. However, at high loads (e.g. 50% to90%), the microparticles were dense. Thus, the invention includesmicroparticles or sustained release devices manufactured by the processof the invention.

The invention also includes an improved sustained release device whichhas incorporated therein an amount of active agent greater than or equalto at least about 50% by weight (w/w) of the polymer-based sustainedrelease device (also referred to as a "high load"). A preferred range isfrom about 50% (w/w) to about 85% (w/w) and more preferably from about50% (w/w) to about 70% (w/w). In general, these high load microparticlesare difficult to manufacture employing the prior art processes and thehigh encapsulation efficiency observed is unexpected. In addition, thehigh load microparticles would not be expected to exhibit improvedsustained release of active agent over lower active agent loads. In aspecific embodiment, the polymer-based sustained release device has ahigh load of azaline B. In a more specific embodiment, the polymer ofthe sustained release device is poly(lactide-co-glycolide) having a highload of azaline B.

In a further embodiment, the improved polymer-based sustained releasedevice has an increased period of sustained release and/or increasedbioavailability over that achieved with a device prepared by a methodwhich does not solubilize the active agent in the polymer solution. Forexample, when microparticles containing azaline B are prepared employingmethylene chloride as the sole polymer solvent the active agent is notsolubilized (referred to herein as the "Particulate Method"). Comparisonof active agent release from these microparticles with those preparedemploying DMSO as the continuous phase (active agent solubilized) can beachieved by comparing FIGS. 1 and 2. Clearly, the polymer-basedsustained release devices prepared by the process wherein the activeagent is solubilized (FIG. 2), demonstrate an increased period ofsustained release over those devices wherein a single polymer solventwhich does not solubilize the active agent is employed (FIG. 1).

Without being bound by a particular theory it is believed that therelease of the biologically active agent can occur by at least twodifferent mechanisms. First, release can occur due to degradation of thepolymer. Second, biologically active agent can be released by diffusionthrough the channels generated in the polymer-based sustained releasedevice, such as by the dissolution of the active agent or by voids orpores created by the removal of the polymer/active agent solvent duringthe synthesis of the drug delivery device.

The rate of degradation can be controlled by changing polymer propertiesthat influence the rate of hydration and/or degradation of the polymer.These properties include, for instance, the ratio of different monomers,such as lactide and glycolide, comprising a polymer; the use of the L-or D-isomer or racemic mixture of a chiral monomer; a polymer, such as apoly(lactide-co-glycolide) polymer that has, for instance, a hydrophobicor a hydrophilic end group; the morphology of the particle as impactedfor example, by choice of solvents for polymer during preparation; andthe molecular weight of the polymer. These properties can affecthydrophilicity and crystallinity, which control the rate of hydration ofthe polymer. Hydrophilic excipients such as salts, carbohydrates andsurfactants can also be incorporated to increase hydration and which canalter the rate of erosion of the polymer.

In addition, the active agent in the sustained release device of thepresent invention can also contain other excipients, such asstabilizers, bulking agents or aggregation-stabilizing agents.Stabilizers are added to maintain the potency of the biologically activeagent during device fabrication, storage and over the duration of theagent's release. Suitable stabilizers include, for example,carbohydrates, amino acids, fatty acids and surfactants which are knownto those skilled in the art. For amino acids, fatty acids andcarbohydrates, such as sucrose, lactose, mannitol, inulin, maltose,dextran and heparin, the mass ratio of carbohydrate to biologicallyactive agent is typically between about 1:10 and about 20:1. Forsurfactants, such as polysorbates (e.g., Tween™) and poloxamers andpoloxamines (e.g., Pluronic™), the mass ratio of surfactant to agent istypically between about 1:1000 and about 1:2.

Aggregation-stabilizing agents are agents which stabilize thebiologically active agent against significant aggregation in vivo overthe sustained release period. Typically an aggregation stabilizerreduces the solubility of the biologically active agent, precipitatesout a salt of the agent or forms a complex of the agent. The aggregationstabilizer and the biologically active agent can be separately containedwithin the drug delivery device, such as a device containing particlesof aggregation stabilizer and separate particles of biologically activeagent, and/or can be combined together in complexes or particles whichcontain both the aggregation stabilizer and the biologically activeagent.

The use of aggregation-stabilizing agents is also described inco-pending U.S. patent application Ser, Nos. 08/478,502 and 08/483,318both filed on Jun. 7, 1995, and U.S. patent application Ser. No.08/521,744, filed on Aug. 31, 1995 the teachings of which areincorporated herein by reference in their entirety.

Metal cations can be suitable as aggregation-stabilizing agents. Thesemetal cations include cations of transition metals, such as Zn⁺², Cu⁺²,Co⁺², Fe⁺³ and Ni⁺². The use of metal cations as aggregation-stabilizingagents, is also described in co-pending U.S. patent application Ser. No.08/279,784, filed Jul. 25, 1994, co-pending U.S. patent application Ser.No. 08/521,744, filed Aug. 31, 1995, PCT Patent ApplicationPCT/US95/07348, filed Jun. 7, 1995, U.S. Pat. No. 5,654,010 issued toJohnson et al. and U.S. Pat. No. 5,667,800 issued to Johnson et al., theteachings of which are incorporated herein by reference in theirentirety.

The polymer-based sustained release device can also contain a metalcation component which is dispersed within the polymer. This metalcation component acts to modulate the release of biologically activeagent from the polymeric matrix.

A metal cation component used in modulating release typically containsat least one type of multivalent metal cation. Examples of metal cationcomponents suitable to modulate release of biologically active agent,include, or contain, for instance, Mg(OH)₂, MgCO₃ (such as4MgCO₃.Mg(OH)₂ ·5H₂ O), ZnCO₃ (such as 3Zn(OH)₂ ·2ZnCO₃), CaCO₃, Zn₃ (C₆H₅ O₇)₂, Mg(OAc)₂, MgSO₄, Zn(OAc)₂, ZnSO₄, ZnCl₂, MgCl₂ and Mg₃ (C₆ H₅O₇)₂. A suitable ratio of metal cation component-to-device is betweenabout 1:99 to about 1:1 by weight. The optimum ratio depends upon thepolymer and the metal cation utilized.

A polymeric matrix containing a dispersed metal cation component tomodulate the release of a biologically active agent from the polymericmatrix is further described in U.S. Pat. No. 5,656,297 issued toBernstein et al. and co-pending PCT Patent Application PCT/US95/05511,the teachings of which are incorporated herein by reference in theirentirety.

In a third aspect, the present invention provides a method of using thepolymer-based sustained release device comprising providing a sustaineddelivery rate of active agent, in a subject, over a therapeuticallyuseful period of time, by administering to the subject a dose of saidpolymer-based sustained release device.

The sustained release device of this invention can be administered to ahuman, or other animal, by injection, implantation (e.g, subcutaneously,intramuscularly, intraperitoneally, intracranially, intraocularly,intravaginally and intradermally), administration to mucosal membranes(e.g., intranasally or by means of a suppository), or in situ delivery(e.g. by enema or aerosol spray) to provide the desired dosage of anagent based on the known parameters for treatment with that agent of thevarious medical conditions.

Even though the invention has been described with a certain degree ofparticularity, it is evident that many alternatives, modifications, andvariations will be apparent to those skilled in the art in light of theforegoing disclosure. Accordingly, it is intended that all suchalternatives, modifications, and variations which fall within the spiritand scope of the invention be embraced by the defined claims. Theinvention will now be further and specifically described by thefollowing examples.

Exemplification Methods

The polymers employed in the following examples are described below:

Purchased from Boehringer Ingelheim

RG 502H: 10k MW, 50:50 Poly(D,L-lactide-co-glycolide) (PLGA),hydrophilic end groups

RG 501H: 5k MW, 50:50 Poly(D,L-lactide-co-glycolide) (PLGA), hydrophilicend groups

R 104: 5k MW, Poly(D,L-lactide)

Purchased from Birmingham Polymers, Inc., Birmingham Ala.

Lot 112-43-1: 5k MW, Poly(D,L-lactic acid)

EXAMPLE 1 Polymer Solvent Method

A polymer/active agent solution can be formed by dissolving anappropriate amount of polymer and active agent in a continuous phasecomprising one or more polymer solvents which also solubilize the activeagent. If more than one polymer solvent is employed both need notsolubilize the active agent. The polymer/active agent solution can thenbe atomized into droplets which can be frozen. The solvent is thenremoved from the frozen droplets to form a polymer/active agent matrixby diffusion of the polymer solvent into a polymer non-solvent phase,the cure phase. The cure phase can be comprised of a single solvent or amixture of solvents. The particles are collected from the polymernon-solvent by filtration and residual polymer solvent and non-solventare removed by evaporation. The dry product is sieved through anappropriately sized mesh so as to produce an injectable product.

The process can be summarized as follows:

Formation of a polymer/active agent solution by dissolving PLGAcopolymer (3-28% (w/v)) and active agent in DMSO.

Atomization of the polymer active agent solution by sonication, andfreezing of the droplets by contact with liquid nitrogen.

Extraction of the polymer/active agent solvent in 80° C. ethanol curesolvent, thereby forming a polymer active agent matrix.

Isolation of the particles from the cure solvent by filtration.

Removal of remaining solvents by evaporation.

Sizing of particles by passage through an appropriately sized mesh so asto produce an injectable product.

The "Particulate Method," referred to in FIGS. 1 and 3, is a processsimilar to that summarized above, but where the active agent is added tothe polymer solution as a solid and remains in the solid or particulateform (i.e., does not dissolve) throughout the process.

EXAMPLE 1.1 63% (w/w) Peptide Loaded, PLGA Microparticled, Ethanol CurePhase

High load microparticles comprising PLGA and azaline B were prepared asfollows:

1) A solution comprising DMSO (0.701 ml), PLGA (0.043 g) (10k MW,hydrophilic end groups) and azaline B acetate (0.185 g) was prepared bymixing the components at room temperature.

2) The solution from step 1 was atomized by an ultrasonic atomizingprobe (Sonics & Materials #630-0507) at a constant flow rate of 0.3ml/minute.

3) The atomized droplets were frozen upon passage through a coldnitrogen gas phase and then into liquid nitrogen. The liquid nitrogenlayer was placed over a frozen non-solvent phase (100% ethanol).

4) The liquid nitrogen layer containing the frozen droplets was allowedto evaporate at -80° C. and the polymer/active agent solvent (DMSO) wasextracted from the frozen droplets over an 18 hour incubation time at-80° C. employing ethanol as the cure phase.

5) The microparticles were separated from the cure phase by filtrationand freeze-dried.

6) The dry product was sieved through a 180 μm mesh sieve.

EXAMPLE 1.2 63% (w/w) Peptide Loaded, PLGA Microparticles,Heptane/Ethanol Cure Phase

1) A solution comprising DMSO (50.0 ml) and PLGA (5.0 g)(10k MW,hydrophilic end groups) copolymer was prepared at room temperature. To3.0 ml of the polymer solution was added 0.233 g of azaline B acetate,and allowed to dissolve.

2) The solution from step 1 was atomized by an ultrasonic atomizingprobe (Sonics & Materials #630-0507) at a constant flow rate of 0.3ml/minute.

3) The atomized droplets were frozen upon passage through a coldnitrogen gas phase and then into liquid nitrogen. The liquid nitrogenlayer was placed over a frozen non-solvent phase (75% heptane; 25%ethanol, v/v).

4) The liquid nitrogen layer containing the frozen droplets was allowedto evaporate at -80° C. The frozen non-solvent phase was allowed to meltat -80° C. and the polymer/active agent solvent (DMSO) was extractedfrom the frozen droplets over an 18 hour incubation time at -80° C.employing a mixture of heptane/ethanol (75:25) as the cure phase.

5) The microparticles were separated from the cure phase by filtrationand freeze-dried.

6) The dry product was sieved through a 180 μm mesh sieve.

Microparticles containing a 49% and a 41% load of azaline B (FIG. 3)were also prepared employing the process of Example 1.1.

EXAMPLE 2 Polymer Solvent/Polymer Non-solvent

The general procedure for the formation of microparticles using amixture of polymer solvent/polymer non-solvent, is similar to thePolymer Solvent Method, described above, with the exception that thecontinuous phase comprises a polymer solvent/polymer non-solventmixture. This method provides for the solubilization of active agentssuch as tRNA and ovalbumin, which are not readily soluble in polymersolvents. In the example described below the polymer non-solventemployed was water.

The polymer solvent/water mixture was formulated such that the additionof water to the system increased the solubility of the active agent, butdid not exceed the concentration at which substantial precipitation ofthe polymer would result. In addition, the polymer non-solvent can beused to predissolve the active agent; the resulting solution can then beadded to the polymer solution such that a transient continuous phaseresults. The transient continuous phase can be further processed priorto precipitation of the active agent or polymer.

A specific example of this method is the manufacture of a devicecomprising D,L-PLA (100% D,L-Poly(lactic acid), 5k MW) and ovalbumin ata 1% (w/w) load.

1) A 5% (w/v) D,L-PLA solution was prepared by dissolving D,L-PLA inDMSO at 50 mg D,L-PLA per ml of DMSO.

2) The active agent ovalbumin was dissolved in deionized water at aconcentration of 100 mg/ml. 20 microliters of the aqueous solution wasadded to 39.6 ml of the polymer solution in a dropwise manner, withmixing.

3) The solution from step 2 was atomized by an air atomization. Theatomized droplets are collected in a -70° C. cure phase (ethanol),resulting in formation of the polymer matrix, with drug distributedthroughout.

4) The microparticles were separated from the cure phase by filtrationand freeze-dried.

5) The dry product was sieved through a 180 μm mesh sieve.

EXAMPLE 3 Polymer Solvent/Polymer Non-solvent

The general procedure for the formation of microparticles using amixture of a polymer solvent and a polymer non-solvent, is similar tothe Polymer Solvent Method, described in detail above, with theexception that the continuous phase is comprised of a polymer solventand a polymer non-solvent, for example, DMSO and ethanol. It isunderstood that the polymer non-solvent in this example is also anactive agent non-solvent.

A specific example of this method is the manufacture of a sustainedrelease device comprising PLGA and azaline B acetate at a 60% (w/w)load.

1) A 10% (w/v) solution of PLGA copolymer (10k MW, hydrophilic endgroups) in a mixture of DMSO/ethanol (75:25 v/v) was prepared.

2) Azaline B acetate, in dry powder form was dissolved in the polymersolution at approximately room temperature to give a final concentrationof 0.233g of azaline B acetate per ml of polymer solution.

3) The solution from step 2 was atomized by an ultrasonic atomizingprobe (Sonics & Materials #630-0507) at a constant flow rate of 0.3ml/minute.

4) The atomized droplets were frozen upon passage through a coldnitrogen gas phase and then into liquid nitrogen. The liquid nitrogenlayer was placed over a frozen non-solvent phase (100% ethanol).

5) The liquid nitrogen layer containing the frozen droplets was allowedto evaporate at -80° C. The frozen non-solvent phase was allowed to meltat -80° C. and the polymer solvent/active agent non-solvent(DMSO/ethanol) was extracted from the frozen droplets over an 18 hourincubation time at -80° C. employing ethanol as the cure phase.

6) The microparticles were separated from the cure phase by filtrationand freeze-dried.

7) The dry product was sieved through a 180 μm mesh sieve.

Microparticles containing a 54% and a 68% load of azaline B (depicted inFIG. 2) were also prepared employing this process.

EXAMPLE 4 Polymer Solvent/Active Agent Non-solvent (Olive Oil CurePhase)

The general procedure for the formation of microparticles using amixture of polymer solvent/active agent non-solvent, is similar to thePolymer Solvent Method, described in detail above, with the exceptionthat the continuous phase comprises a polymer solvent/active agentnon-solvent.

A specific example of this method, is the preparation of a sustainedrelease device comprising PLGA and azaline B at a load 70% (w/w) activeagent.

1) A 10% (w/v) solution of PLGA copolymer (10k MW, hydrophilic endgroups) in a mixture of DMSO/acetone (80:20 v/v) was prepared.

2) Azaline B acetate, in dry powder form was dissolved in the polymersolution at room temperature to give a final concentration of 0.233 g ofazaline B per ml of polymer solution.

3) The solution resulting from step 2 was atomized by an ultrasonicatomizing probe (Sonics & Materials #630-0507) at a constant flow rateof 0.3 ml/minute.

4) The atomized droplets were frozen upon contact with cold (4° C.)olive oil.

5) The polymer solvent/active agent non-solvent (DMSO/acetone) wasextracted from the frozen droplets over a 7 day incubation time at 4°C., with mixing.

6) The microparticles were separated from the oil by the formation of anemulsion in which the oil phase containing the microspheres was rapidlymixed with a 4×volume of a heptane/ethanol mixture (75:25 v/v). Themicroparticles were separated from the emulsion phase by filtration. Theemulsion/filtration procedure was repeated 3 times.

7) The microparticles were separated from the final emulsion phase byfiltration and freeze-dried.

8) The dry product was sieved through a 180 μm mesh sieve.

EXAMPLE 4.1 Polymer Solvent/Active Agent Non-solvent--65% (w/w) Load(Heptane/Ethanol 75:25 Cure Phase)

1) A 10% (w/v) PLGA copolymer solution was prepared by dissolving PLGAcopolymer (10k MW, hydrophilic end groups) in a mixture of DMSO/acetone(80:20 v/v).

2) Azaline B acetate, in dry powder form was dissolved in the polymersolution at room temperature to give a final concentration of 0.233 g ofazaline B per ml of polymer solution.

3) The solution resulting from step 2 was atomized by an ultrasonicatomizing probe (Sonics & Materials #630-0507) at a constant flow rateof 0.3 ml/minute.

4) The atomized droplets were frozen upon passage through a coldnitrogen gas phase and then into liquid nitrogen. The liquid nitrogenlayer was placed over a frozen non-solvent phase (75:25% v/vheptane:ethanol).

5) The liquid nitrogen layer containing the frozen droplets was allowedto evaporate at -80° C. The frozen non-solvent phase was allowed to meltat -80° C. and the polymer solvent/active agent non-solvent(DMSO/acetone) was extracted from the frozen droplets over an 18 hourincubation time at -80° C. employing a mixture of heptane/ethanol(75:25) as the cure phase.

6) The microparticles were separated from the non-solvent phase byfiltration and freeze-dried.

7) The dry product was sieved through a 180 μm mesh sieve.

EXAMPLE 4.2 Polymer Solvent/Active Agent Non-solvent--68%

(w/w) Load (Ethanol Cure Phase)

1) A 10% (w/v) PLGA copolymer solution was prepared by dissolving PLGAcopolymer (10k MW, hydrophilic end groups) in a mixture of DMSO/acetone(80:20 v/v).

2) Azaline B acetate, in dry powder form was dissolved in the polymersolution at room temperature to give a final concentration of 0.233 g ofazaline B per ml of polymer solution.

3) The solution resulting from step 2 was atomized by an ultrasonicatomizing probe (Sonics & Materials #630-0507) at a constant flow rateof 0.3 ml/minute.

4) The atomized droplets were frozen upon passage through a coldnitrogen gas phase and then into liquid nitrogen. The liquid nitrogenlayer was placed over a frozen non-solvent phase (ethanol).

5) The liquid nitrogen layer containing the frozen droplets was allowedto evaporate at -80° C. The frozen non-solvent phase was allowed to meltat -80° C. and the polymer solvent/active agent non-solvent(DMSO/acetone) was extracted from the frozen droplets over an 18 hourincubation time at -80° C. employing ethanol as the cure phase.

6) The microparticles were separated from the non-solvent phase byfiltration and freeze-dried.

7) The dry product was broken up by a gentle, manual grinding and passedthrough a 180 μm mesh sieve.

EXAMPLE 5 Manufacture of Sterile Product Using the "MicroparticulateMethod"--Polymer Solvent/Active Agent Non-solvent

The method can be used to produce sterile product by enabling thesterile filtration (0.2 μm ) of the polymer/active agent solution, priorto further processing.

For example, the manufacture of a sterile 15% (w/w) loaded azaline Bacetate/PLGA formulation was performed as follows:

1) A 20% (w/v) solution of PLGA copolymer (10k MW, hydrophilic endgroups) in dichloromethane was prepared by dissolving 0.2 g of PLGA perml of dichloromethane. The polymer solution (639 ml) was introduced intoa sterile vessel, equipped with a rotor-stator homogenizer, using 0.22μm filtration. The polymer solution was chilled to approximately -77° C.

2) Azaline B was dissolved in DMSO at a concentration of 12.5% (w/w) bydissolving 0.125 g of azaline B per gram of DMSO.

3) 135 g of the azaline B solution was introduced slowly into thesterile tank containing the polymer solution via 0.22 μm filtration. Therotor-stator homogenizer was run immersed in the polymer solution andthe temperature was maintained at approximately -77° C. during theaddition of the azaline B/DMSO solution.

4) The DMSO/azaline B solution freezes upon introduction to the coldpolymer solution and is dispersed throughout the dichloromethane polymersolution by action of the rotor-stator homogenizer. As the DMSO anddichloromethane mix, peptide precipitates as a microsuspension.

5) The resulting microsuspension was atomized by air atomization and thedroplets frozen by contact with liquid nitrogen.

6) The frozen droplets were mixed with an excess volume of ethanol uponwhich the DMSO and the dichloromethane were extracted to produce apolymer matrix with the active agent dispersed throughout.

The following Table summarizes the PLGA microparticles, preparedaccording to Examples 1 through 5. % Load and Encapsulation Efficiencywere determined by analysis of the nitrogen content of themicroparticles using a CE-440 Elemental Analyzer available from ExeterAnalytical, Inc., Lowell, Ma.

    __________________________________________________________________________         Polymer/                                                                                                           Encapsulation                                                                          Efficiency                 Example                                                                                       Process                                                                                 Cure Phase                                                                             Product                                                                              (% w/w)                                                                             (% Target)                    __________________________________________________________________________    1.1  DMSO Atomization                                                                         Liquid                                                                             -80° C. Ethanol                                                                Microparticles                                                                       63    90                                                                 Nitrogen                                       1.2            Atomization                                                                         Liquid                                                                            -80° C.                                                                    Microparticles                                                                          63            90                                              Heptane/Ethanoln                                                                      75:25                                          2             Atomization                                                                          Ethanol                                                                          -80° C. Ethanol                                                             Microparticles                                                                        1           67                                           Water                                                                                         (<0° C.)                               3             Atomization                                                                          Liquid                                                                            -80° C. Ethanol                                                            Microparticles                                                                       60          85                                            Ethanol                                                                                       Nitrogen                                      4             Atomization                                                                          Liquid                                                                            4° C. Olive Oil                                                            Microparticles                                                                        70           100                                         Acetone                                                                                       Nitrogen                                      4.1           Atomization                                                                          Liquid                                                                            -80° C.                                                                     Microparticles                                                                        65             93                                       Acetone                                                                              Heptane/Ethanolen                                                                     75:25                                          4.2          Atomization                                                                               -80° C. Ethanol                                                            Microparticles                                                                       68           97                                           Acetone                                                                                       Nitrogen                                      5         Atomizationene                                                                           Liquid                                                                            -80° C. Ethanol                                                            Microparticles                                                                       15 (Sterile)                                                                        100                                           "Micro-hloride/                                                                           Nitrogen                                                               particulate"                                                   __________________________________________________________________________

In Vivo Data

EXAMPLE 6: Rat Estrous Model

The Rat Estrous Cyclicity Model is described in detail in Hahn et al.,Biological Assays Utilized to Characterize LHRH and its Analogs. In:LHRH and its Analogs, Contraception and Therapeutic Applications (Eds.Vickery B H, Nestor J J, and Hafez E S E), pp. 49-60. MTP Press Ltd,Lancaster, England, 1984, the contents of which are incorporated hereinby reference. The model is used to assess the pharmacodynamic effectwhich an effective serum level of azaline B (at least 2.0 ng/ml),provided by the administration of a sustained release device of theinvention, has on the estrous cycle of the rat. When the minimumeffective concentration of azaline B is present in serum, the estrusphase of the estrous cycle of the rat is suppressed and the rat remainsin the diestrus phase of the estrous cycle.

Microparticles containing an effective amount of azaline B were preparedaccording to the "Particulate Method," described above, and the methodsof Examples 1 and 3. The presence of azaline B activity was measured asthe percent of a test group which remained in the diestrus phase of theestrous cycle. Animals were subcutaneously injected with equal amountsof azaline B either encapsulated in a microparticle or unencapsulated.The injections employed a vehicle of 3% carboxymethyl cellulose (lowviscosity) and 1% Tween-20 in saline. The results are depicted in FIGS.1 and 2 which show a plot of the percent of animals per treatment groupin diestrus for each formulation evaluated versus time. FIG. 1 show thatrats treated with the particulate formulations, were observed to begincycling after 8-10 days. The rats treated with the microparticlesprepared according to the method of the invention, specifically Examples1 and 3, as shown in FIG. 2 had a further delay in the onset of cycling,indicating the presence of an effective serum concentration for a longerperiod of time.

EXAMPLE 7 Evaluation of Azaline B Serum Levels

Microparticles were processed using the "Particulate Method" describedabove and the methods of Examples 1 and 3. Animals were subcutaneouslyinjected with equal amounts of azaline B either encapsulated in amicroparticle or unencapsulated. The injections employed a vehicle of 3%carboxymethyl cellulose (low viscosity) and 1% Tween-20 in saline. Serumlevels (ng/ml) were determined at various times over a 28 day period andthe results are show in FIG. 3 as a plot of the concentration of azalineB (ng/ml), versus time. Serum levels were determined using anelectrochemiluminescent immunoassay method. In this method quantitationis performed using an antibody that is specific for azaline B, andconcentration is determined by comparison to a standard curve.

Briefly, the animals were anesthetized with halothane and blood sampleswere collected via a lateral tail vein. The blood was clotted at roomtemperature, centrifuged at approximately 6000×g for about five minutesand stored at -70° C. until analysis could be performed.

FIG. 3 is a graph of the serum levels of azaline B versus time. TheFigure demonstrates that the release profile of high load microparticlesprepared according the methods of Examples 1 and 3 was improved overthat seen with microparticles prepared according to the "ParticulateMethod."

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A method of forming a polymer-based sustainedrelease device comprising the steps of:a) forming a polymer/biologicallyactive agent solution by mixing a polymer, a continuous phase comprisingone or more polymer solvents and a biologically active agent wherein thepolymer and biologically active agent are present in relativeconcentrations such that the device contains about 50% by weight or moreof the biologically active agent; and b) removing the continuous phaseof step (a) thereby forming a solid polymer/biologically active agentmatrix.
 2. The method of claim 1 wherein the continuous phase comprisesDMSO.
 3. The method of claim 1 wherein the biologically active agent isa peptide or an antigen.
 4. The method of claim 1 wherein thebiologically active agent is an LHRH analog.
 5. The method of claim 1wherein the biologically active agent is azaline B.
 6. The method ofclaim 1 wherein the polymer is selected from the group consisting of:poly(lactide)s, poly(glycolide)s, poly(lactide-co-glycolide)s,poly(lactic acid)s, poly(glycolic acid)s, poly(lactic acid-co-glycolicacid)s, blends thereof, and copolymers thereof.
 7. A method for forminga polymer-based sustained release device comprising the steps of:a)forming a polymer/biologically active agent solution by mixing apolymer, a continuous phase comprising one or more polymer solvents anda biologically active agent wherein the polymer and biologically activeagent are present in relative concentrations such that the devicecontains about 50% by weight or more of the biologically active agent;b) forming droplets of the polymer/biologically active agent solution;and c) removing the continuous phase of step (a) from thepolymer/biologically active agent solution thereby forming a solidpolymer/biologically active agent matrix.
 8. The method of claim 7wherein the continuous phase comprises DMSO.
 9. The method of claim 8further comprising the step of freezing the droplets of thepolymer/active agent solution prior to removing the continuous phasewherein the continuous phsae is removed by extraction.
 10. The method ofclaim 7 wherein the continuous phase is removed by evaporation.
 11. Themethod of claim 7 wherein the droplets are microdroplets.
 12. The methodof claim 7 wherein the biologically active agent is a peptide or anantigen.
 13. The method of claim 7 wherein the biologically active agentis an LHRH analog.
 14. The method of claim 7 wherein the biologicallyactive agent is azaline B.
 15. The method of claim 7 wherein the polymeris selected from the group consisting of: poly(laclide)s,poly(glycolide)s, poly(lactide-co-glycolidc)s, poly(lactic acid)s,poly(glycolic acid)s, poly(lactic acid-co-glycolic acid)s, blendsthereof, and copolymers thereof.